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Direct voltammetric determination of ascorbic acid in

natural paprika fruits without sample pretreatment

Journal: Electroanalysis

Manuscript ID: elan.201400603.R1

Wiley - Manuscript type: Full Paper

Date Submitted by the Author: n/a

Complete List of Authors: Őri, Zsuzsanna Emese; University of Pécs, General and Physical Chemistry Nagy, Livia; University of Pécs, Szentágothai János Research Center Kiss, László; University of Pécs, Szentágothai János Research Center Kovács, Barna; University of Pécs, Department of General and Physical Chemistry Nagy, Géza; University of Pécs, General and Physical Chemistry

Keywords: Ascorbic acid, Paprika, Tortuosity, Vitamins, Voltammetry

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Direct voltammetric determination of ascorbic acid in natural paprika fruits without sample pretreatment

Zsuzsanna Őri,a,b Lívia Nagy,a László Kiss,a Barna Kovács,b Géza Nagy b* a University of Pécs, Szentágothai János Research Center, Pécs, Ifjúság útja 20, 7624, Hungary b University of Pécs, Department of General and Physical Chemistry, Ifjúság útja 6, H-7624 Pécs, Hungary * e-mail: g-nagy@gamma.ttk.pte.hu

Received: ((will be filled in by the editorial sttaff)) Accepted: ((will be filled in by the editorial sttaff))

Abstract Direct electrochemical measuring technique for estimation the ascorbic acid (AA) content of freshly cut yellow pepper fruits without special sample treatment was worked out. Dialysis membrane modified glassy carbon electrode combined with short time chronoamperometric measuring method was employed to eliminate the interfering effect of tortuosity and electrode fouling. Viscosity change inside the diffusion layer was taken in account. AA content of the fruit was found to be 114.5 mg/ 100g, and it is shown that the metal ions derived from iron blade are able to accelerate the oxidation process significantly. Classical iodometric titration was applied as a reference method.

Keywords: Ascorbic acid, Paprika, Tortuosity, Vitamins, Voltammetry

DOI: 10.1002/elan.((will be filled in by the editorial sttaff))

1. Introduction

Ascorbic acid (AA) is a small-molecular-weight, water-soluble antioxidant vitamin that participates in many biochemical reactions [1] and serves as a quality indicator of foods and drinks [2]. The quick monitoring of vitamin C levels during food production and quality control stages is an important analytical task [3]. Electrochemical techniques are often preferred to laborious instrumental methods [4, 5, 6, 7, 8] in AA assessment due to the simplicity of the procedure and of the instrumentation, to the fast response, and low cost. [9] Ascorbic acid can be easily oxidized to dehydroascorbic acid (DHA), and this redox reaction is used for its electrochemical determination [10].

Several examples of viable AA determination in various media by bare electrodes can be found in the literature, even in the presence of interferents. These methods include sample pretreatment like centrifugation [11, 12] or filtration and dilution [13] in case of fruit juices. The build-up of oxidation products can diminish the electrode response, requiring lengthy electrode cleaning procedures between each measurements [14]. PEDOT conducting polymer modified gold electrode was prepared by Türke et al.

[14], and tested by CV measurements. The peak related to the AA oxidation was obtained at 240 mV potential, was well separated from the main polyphenol oxidation peak in model wine however, repetitive analysis of AA determination in real sample with the same electrode was not carried out [14]. Thin films of polyaniline containing dopant ions polyvinylsulfonate (PVS) and polystyrenesulfonate (PSS) have been prepared on Pt disk microelectrodes and tested for AA oxidation. A potential of 0.1 V (v.s. SCE) was found to be appropriate for the amperometric determination of AA in beverages with minimal interference from other oxidisable compounds. However, exposure to wine and orange juice led to a diminution of the sensor response. The electrode fouling was due to adsorption of some beverage components [15]. A promising solution for the elimination of the electrode fouling and interference was presented for an electroactive anesthetic drug, propofol, determination in a complex medium. The method employs a PVC membrane coated GC working electrode [16, 17].

“Built-in diffusion layer” modified GC working electrode with chronoamperometric detection was developed recently for analyte determination in tortuous medium [18]. If the sample is a tortuous

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suspension containing particulate matter, live or fragmented tissue , pulp, etc., the mass transport of electroactive species in it is hindered [19]. In voltammetric analysis, for assessing the electroactive analyte concentration, the current value taken in sample solution is compared to current values recorded in calibrating standards. The voltammetric current depends on the mass transport characteristics of the analyte in the investigated sample matrix. Therefore, in order to get reliable sample concentration value, the mass transport properties must be identical in the calibrating solutions and in real samples. As long as the diffusion profile of the analyte is inside of the built-in diffusion layer, the calibrating chronoamperometric transients can be used for evaluating transients (at each identical time intervals) taken in tortuous media due to the fact that the sample tortuosity has no effect on diffusion coefficient of the analyte in the built-in diffusion layer. It is identical in both cases regardless the difference in tortuosity of the media. The applicability of the in situ analytical method was tested for determination of active chlorine concentration in different bleaching powder treated soil samples [20].

Capsicum species (pepper/paprika) are grown all over the world, primarily in tropical and subtropical countries and the fruits are an excellent sources of health-related compounds, such as ascorbic acid (vitamin C), carotenoids, tocopherols, capsaicinoids and flavonoids [21]. A comprehensive characterization of the phenolic and other polar compounds were performed by HPLC-MS method in pepper samples and besides the two identified isomers of ascorbic acid, 43 further compounds were found. Among them, AA has almost the smallest molecular weight [22].

Recently, a Screen-Printed Carbon electrode modified with Nylon-6 nanofibers membrane was successfully applied for amperometric determination of AA in fruits without sample treatment. The membrane significantly improved the selectivity towards AA, and entailed a widening of the linearity range [23]. In our study, a dialysis membrane modified GC electrode with short time chronoamperometric detection is applied for the reagentless, in situ assessment of the AA concentration in paprika fruit without sample pretreatment. A resting period with opened circuit prior to the measurement enables the equilibration of the membrane layer by the analyte, and the depletion of the remaining oxidation products. Due to the appropriately short time detection the tortuosity does not affect the result–as the diffusion profile is still inside the membrane layer during the

measurement-, and there is a minimal possibility for the electrode fouling. Therefore the presented method is presumed to possess further advantages in comparison to the methods employing conventional amperometric detection technique.

Traces of iron can get into the fruit tissue during food processing. Using our method the catalytic effect of this can be detected. The extent of the autooxidation depends on the nature of the cutting blade used for slicing the fruit tissue.

2. Experimental

Chemicals and materials

Ascorbic acid (AA), sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, glycerol, potassium iodide, iodine, soluble starch used were of analytical reagents grade and were purchased from VWR BDH Prolabo; while iodine and potassium chloride were obtained from Reanal (Hungary).

Phosphate buffer made with double deionized water and containing 0.5M KCl served as solvent for AA solutions and was used as background electrolyte for measurements in aqueous solutions. The pH of the buffer was adjusted to 5.9 in order to match the natural pH of the paprika juice. It worth mentioning that at low pH AA is fully protonated and is relatively stable. Its maximal stability was found in the pH range of 4–6 [24]. AA concentration of different solution is expressed throughout the text in mg/100 g unit in accordance with the generally applied practice in food sciences. AA solutions were prepared each time freshly before use. In all cases -except for the direct measurements in the paprika tissue- dissolved oxygen was purged out from the cell by bubbling high purity nitrogen for 10 min before the experiment.

Glassy carbon (GC) rod for electrode preparation was purchased from VWR International Ltd. Buehler (USA). MicroPolish alumina powder of different particles size and Buehler MicroCloth (No. 40–7218) were used in wet electrode polishing. The dialysis membrane (Technikon TM Pre-mount type “C”) used as size-exclusion diffusion layer was taken from AutoAnalyzer accessories (Technicon Instruments Corporation) with a thickness of 127 µm; pore diameters ranged between 4 and 6 nm.

Yellow sweet pepper (Capsicum annuum Linn.) was purchased from the local supermarket, and it was washed under tap water. For juice preparation the peppers were masticated with a plastic rasp and the

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obtained liquid was separated filtering it through a plastic kitchen strainer. In case of making pepper slice samples ceramic-, stainless steel- or iron blade was used to break up cells slightly on the surface of the paprika tissue. The blades were in contact with the paprika tissue for about 2-3 minutes.

For viscosity measurements paprika juice was centrifuged with 10.000 rpm for 5 min then the transparent aliquot was filtered with a 0.2 micron Injection Filter (VWR BDH Prolabo). AA solutions of different viscosity were made mixing buffer solution and glycerol in different ratio as follows: 150mg AA dissolved in 10 ml buffer; 150 mg AA in 9.75ml buffer + 0.25ml glycerol; 150 mg AA in 9.5ml buffer + 0.5ml glycerol and the same amount of solute in 8.75ml buffer + 1.25ml glycerol.

The iodine reagent used in titrations was made dissolving 2 g potassium iodide and 1.3 g iodine in 1dm3 water.

Electrodes

Homemade GC disc electrode (3 mm in diameter) was used as working electrode sealed in cylindrical Teflon body, a Pt plate as counter electrode (∼4 cm2) and Saturated calomel electrode (SCE) as reference one were employed. The surface of the GC electrode was wet polished with 1, 0.3, and finally, 0.05-µm particle size alumina polishing powder, washed and sonicated for 1 min between and after each polishing steps. Diffusion layer modified GC electrode was made by coating the measuring surface of a freshly polished wet GC electrode with a dialysis membrane cup. It was fixed using a thread and great care was taken to avoid air bubbles under the membrane.

Instruments and electrochemical measurements

In the cyclic voltammetric and chronoamperometric studies, conventional beaker-type measuring cell was applied, except when the chronoamperometric measurements carried out directly on paprika slices. In all measurements CHI 760C electrochemical workstation (CH Instruments, Inc. USA) was employed.

After each chronoamperogram recording the electric circuit was opened to allow time to reach concentration equilibrium between the sample and the diffusion layer. The required time for this equilibration process was determined experimentally. Sampling interval was set to 0.01 s.

Appropriate electrode potential was selected by recording linear sweep voltammograms previously. All measurements were carried out at room temperature. All electrode potentials were measured vs. saturated calomel reference electrode (SCE). The chronoamperometric measurements were performed at 0.6 V constant potential vs. the reference.

Viscosity measurements were carried out at controlled temperature (22 oC) with the application of a Capillary Viscosimeter (Cannon-Fenske Routine, Fungilab ASTM D-445) and the liquid density was determined with a Pycnometer (4AJ-9277400).

Direct electrochemical measurement in paprika

In these experiments the yellow paprika fruits were cut to slices, the surface of the internal side of the slices were slightly ruptured by blades, in 1-2 millimeters depth. The three electrodes were held by one electrode positioner, keeping constantly 2-3 mm distances between the electrode bodies. The working electrode was placed between the counter and the reference one. Before the measurements the electrodes were slowly brought in touch with the wet surface making sure that the membrane of the working electrode is not compressed by the weight of the electrode so the porosity of it is not changed. The counter electrode was gently pricked into the tissue. The scheme of the direct measurement in the fruit is shown in Fig. 1.

Fig. 1. Scheme of the direct measurement carried out in paprika slice. WE: dialysis membrane coated GC electrode, CE: Platinum sheet counter electrode, RE: Saturated calomel electrode.

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2. Results and discussion

As it is well known AA is electroactive. In buffered solution it gives well formed voltammograms using different electrodes and measuring techniques. Assessment of AA concentration in aqueous solution can be done quite easily employing voltammetric methods. The juice of fruits or vegetables can be good background electrolyte for voltammetric measurements owing to their relatively high electrolyte concentration and more or less buffered pH. Therefore it was expected that AA content of vegetables or fruits could be estimated recording voltammograms directly with a special working electrode poked inside their tissue or immersed into the juice prepared by simply crushing their tissue.

Trying to work out voltammetric method for measuring AA in yellow pepper directly, without sample pretreatment or separation step we prepared juice from paprika samples and recorded cyclic voltammograms one after the other in quiescent solution with 0.1 V/s scanning rate using GC working electrode. Between the voltammograms 60 s was the waiting period. Well-formed peak shaped voltammograms were obtained indicating that the juice contained AA. Similar measurements were made in buffered AA solution containing AA in concentration of 100mg/100g. The peak heights (ip) of the voltammograms were measured. The first one was disregarded but the others were normalized to the ip of the second voltammogram (ip2). The obtained ip/ip2. ratios were plotted against the serial number of the sequential voltammograms. The same experiments were made with dialysis membrane coated electrode. The obtained dependences are shown in Fig. 2.

Fig. 2. Normalized peak currents of sequentially recorded cyclic voltammograms recorded with (■) bare GC and with (●) membrane coated GC electrode in buffered AA solution (100mg/ 100g) Normalized peak currents of sequentially recorded cyclic voltammograms recorded with (▲) bare GC and with (▼) membrane coated GC electrode in paprika juice. Potential window: 0-0.9V vs. SCE, Scanning rate: 0.1V/s. Quiet time before and between the CV-s was 60 seconds.

It can be seen that that the peak heights of the

voltammograms taken in buffered AA solution are almost the same. In the tested juice, however the height of the voltammetric peaks recorded with the bare electrode steadily decreases. This indicates the fouling of the electrode. Some ingredient of the juice upon taking part in electrode reaction decreases steadily the activity of the glassy carbon surface. The tenth ip obtained in paprika juice medium is smaller than 75% of the second one. This sensitivity loss caused by electrode fouling seriously obstructs the application of direct voltammetric measurement for quantification of AA content of the juice. Much less electrode fouling could be observed when the voltammograms were made with the membrane coated electrode. Most likely the dialysis membrane acts like a size exclusion layer. It decreases the transport of the harmful species to the electrode surface.

In case of cyclic voltammetric measurements the oxidizing electrode potential (range) is applied for relatively long time periods. Long-time oxidizing potential exposure results in extensive fouling. Applying short time chronoamperometric measurements at the appropriate electrode potential the measuring time can be shortened drastically. We can expect that using dialysis membrane coated electrode and short time measurement with chronoamperometric technique the interference with direct AA assessments in pepper juice caused by electrode fouling can be dramatically decreased.

There is another difficulty if working electrode directly poked into the pepper tissue is tried to be used for measurement of AA content of the natural tissue. It is the tortuosity. The voltammetric measurements generally rely on diffusion of the electroactive species. In tortuous tissue the diffusion of the AA is slower than it is in aqueous solution. Therefore the evaluation of the voltammetric data taken in the tissue cannot be done simply using calibration curves obtained in aqueous solution. In case of direct measurement in tissue, the standard addition technique also cannot be employed. In our earlier experiments carried out with sediments [18] it was shown however, that using

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working electrode modified with built in diffusion layer and short time chronoamperometric measurements in tortuous media, reliable concentration values could be obtained for concentration of electroactive species. As long as the diffusion profile created by the electrode reaction stays inside the built in diffusion layer, the calibration data obtained in aqueous solution can be used for evaluating the data taken in the tortuous media.

During chronoamperometric measurements the concentration of the detected species decreases at the electrode surface. If the electrode holds a built in diffusion layer then certain time is needed between measurements for equilibration of the concentrations between the sample and the modifying layer. In order to determine the necessary equilibrating time sequential chronoamps with different equilibrating (resting) times between measurements were recorded in quiescent AA solution with the membrane covered GC electrode. The equilibration period was set to 15, 30, 45 and 90 s long and applied before and between the repetitive measurements. The normalized current values of the short time chronoamps taken at 0.3 seconds after the electrode potential jump referring to the different equilibration time periods are shown in Fig. 3.

Fig. 3. Normalized current values taken at 0.3 s of the sequentially repeated chronamps using different equilibration time periods between measurements. The current values referring to the repetitions are normalized to the current values of the second chronoamps (i / i2). Dialysis membrane coated GC electrode was used and the AA concentration of the buffered solution (pH 5.9) was 100 mg/ 100g. The equilibration period was set to be 15, 30, 45 and 90 s long with opened circuit and applied before and between the repetitive recordings. Symbols indicate the serial number of the sequentially taken i300 ms values.

As it can be seen in Fig. 3, in case of 15 s equilibration time the current value of the tenth recording is only 86% of the second one. It is clearly visible that longer the resting time the points are getting closer to each other. At the equilibration time of 90 seconds the value of the last point is approx. 100% of the second one so the points are very close to each other. In the further experiments 90 seconds resting time was applied for the equilibration in quiescent medium.

From the experimental findings shown before we could see that, if a working electrode modified with a built in diffusion layer and short time chronoamperometric measurements with long enough equilibrating time is employed, then the AA concentration of yellow pepper tissue could be assessed directly without sample pretreatment and separation steps. The interfering effects of sample tortuosity and matrix caused electrode fouling can be reduced to the acceptable level. However there is one more property of the sample that can bring in uncertainty. It is the viscosity.

In chronoamperometry the current depends on the diffusion coefficient of the electroactive species under diffusion mass transport controlled conditions. Diffusion coefficient depends on the dynamic viscosity. Matrix components can penetrate into the diffusion layer and can change the viscosity there. This effect can question the reliability of the calibration data taken in aqueous solutions.

In order to study the potential effect of viscosity change on the short time chronoamperometric current transients model solutions of different viscosity containing 150mg/ 100 ml AA were made. The solvent of these solutions were prepared by mixing PBS buffer and glycerol in the following V/V % ratios: 87.5/12.5; 95/5; 97.5/2.5; 100/0.

Chronoamperometric measurements of AA oxidation recorded in differently viscous solvent mixtures are shown in Fig. 4.

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Fig. 4. Chronoamperometric transients of AA oxidation recorded in solvent mixtures of different viscosity. Model solutions were composed of 150mg / 100 ml AA and solvent of aq. PBS buffer and of glycerol in varied ratio: d- 87.5/12.5; c- 95/5; b- 97.5/2.5; a- 100/0. (V/V % aq. buffer/V/V% glycerol) Measuring potential: 0.6V vs. SCE, Working electrode: bare GC.

As it can be seen in Fig. 4, chronoamps run at gradually lower and lower current values in accordance with the increasing ratio of glycerol in the solvent mixtures.

If the rate determining step of electrode process is the linear diffusion then after an appropriate potential step the chronoamperometric curves are described by the Cottrell equation,

� ������

��

�����

(1)

Where: i is the current; n: number of electrons; F: Faraday constant; A: surface area of the electrode; cj0: initial concentration of the analyte; D: diffusion constant; t: time. And since, the diffusion coefficient should vary with the viscosity, according to the Stokes-Einstein equation,

� ���

� �� (2)

-where: k: Boltzmann constant; T: absolute temperature; a: effective radius of the molecule-, η is the viscosity then linear relation between i and η-1/2

should hold if the effective radius is constant in the studied range of the examined solvent composition. A plot of inorm versus η-1/2 shows the experimental data in

Fig. 5. Inorm values refer to the chronoamps presented in Fig. 5. and are normalized to that of the current values taken in 100% aq. buffered AA solution. The hydrodynamic viscosity values of the model solutions were determined by using a capillary viscometer and a picnometer.

Since the aim of this experiment is to estimate the deviation in the current due to viscosity discrepancy between the aq. AA solution and the penetrating paprika juice fluid, dynamic viscosity of the centrifuged, filtered paprika juice was measured at the same way like in case of the glycerol-buffer model solutions. The obtained value is indicated in Fig. 5. Calculated inorm value for the centrifuged, filtered Paprika juice is concluded from the experimentally obtained η value for this sample.

Fig. 5. Experimentally obtained inorm current values versus the reciprocal square root of the measured η. Model solutions were composed of 150mg/ 100 ml AA and solvent of aq. PBS buffer and of glycerol in varied ratio: d- 87.5/12.5; c- 95/5; b- 97.5/2.5; a- 100/0. (V/V % aq. buffer/V/V% glycerol). Inorm values are the normalized currents to that of the 100% aq. buffered solution. (inorm =(i/ia)x100) Calculated inorm value for the centrifuged, filtered paprika juice is concluded from the experimentally obtained η value for this sample.

As it can be seen in Fig. 5, the points of the model solutions lay on a straight line with an adjusted R2= 0.996 value so the assumption on the basis of the two equations is justified under these experimental conditions.

Regarding the experimentally obtained η= 1.111 mPa*s dynamic viscosity value for the centrifuged, filtered paprika juice compared to that of the of the 100% buffered AA solution (η=1.011mPa*s), on the basis of the justified correlation, the calculated current value for the paprika fluid is estimated to be 96.36% of the 100% aq. buffered AA solution, that means if the effective radius of the analyte molecules is considered

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to be the same in these systems as well, approx. 3.6% current decreasing is expected due to the viscosity differences.

In order to test the analytical performance and applicability of the diffusion layer coated electrode in the paprika juice - keeping the identified parameters: 90 s equilibration time, 0.3 s-long chronoamp measuring period- standard addition experiments were carried out in the unfiltered paprika juice and in 100 mg/ 100 g AA solution in a parallel way.

During the chronoamperometrically followed standard addition experiments, 20-20-µl of 1000 mg AA/ 5 ml PBS buffer stock solution was spiked repeatedly into 10 ml of the sample solution by a Hamilton-syringe. The sample solution was purged for ten minutes by water saturated nitrogen flow prior and between the measurements. Each addition steps resulted in concentration increase of 40 mg/ 100 g (neglecting the dilution by the solvent dose). After a chronoamperometric measurement, the electrode was removed from the solution, the sample was spiked by a dose, then 30 sec stirring was applied and finally the electrode was immersed into the bulk again.

Fig. 6b shows the results for the described series of measurements taken by the membrane modified electrode while Fig. 6a is a plot for presenting the same experiments under the same conditions carried out with an uncoated GC electrode.

Fig. 6. Standard addition experiments carried out parallel in 100mg AA/ 100g PBS solution and in unfiltered Paprika juice. a) chronoamperometric measurements taken by uncoated bare GC electrode b) chronoamperometric measurements taken by the dialysis membrane coated GC electrode.

As a reference method, redox titration using iodine solution was used to compare the results obtained with the new electrochemical method. In this case 10 ml of the sample solution (AA solution or Paprika juice appropriately spiked) was titrated with iodine solution in a conical flask containing about 75 ml of distilled water and 1 ml of starch indicator solution. Comparison of the results of the standard addition experiments are summarized in Table 1.

Table 1. Summary of the results of the standard addition experiments carried out in unfiltered Paprika juice by three different methods: chronoamperometry with bare GC electrode, chronoamperometry with dialysis membrane layer coated electrode and redox titration).

Bare GC,

ampero.

Modified GC,

ampero.

Redox

titration

Linearity Tendentious

deflection Adj.R

2=0.999 Adj.R

2=0.996

Recovery

% ---

89.5

93.1 # 93.9

Original

AA conc.

mg/100g

--- 115.9 115.5

[#] with viscosity correction

The suggested chronoamperometric method comparing to the titration, resulted in quite accurately determined AA value and outstanding linearity. The recovery value of the chronoamperometric method applying the membrane coated electrode is 4.3% lower

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than that of the titration method. This difference exists mainly due to the investigated viscosity difference between the aquaeous solution and the paprika fluid that was calculated to be approx. 3.6%. So in the future experiments a correction factor is recommended in the evaluation of the chronoamperometric measurements with the membrane coated electrode carried out in the same matrix. The remaining 6-7% recovery loss probably reflects the intrinsic matrix effect of the unfiltered paprika juice.

The method worked out was used for determination of AA in yellow pepper slices prepared with different knifes. In these studies the intact yellow pepper fruits were cut to slices by three different knifes, namely with ceramic, stainless steel and old style iron blade. The surface of internal side of the sample slices were slightly ruptured by the blades. They were kept in humid atmosphere in a beaker closed with watch glass. After a while the electrodes were brought in touch with the wet surface of the slice as it is shown in Fig 1, and upon proper equilibrium time (90s) chronoamps were recorded and the AA concentration was estimated using calibration data taken in aqueous solutions.

Table 2. The analytical results indicating the rate of oxidation of AA content of differently prepared yellow pepper slices. ∆t value indicates the time periods between the application of the blades and the measurements.

Ceramic

blade

∆t=0-10

min

Ceramic

blade

∆t=60-80

min

S. steel

blade

∆t=60-80

Min

Iron

blade

∆t=60-80

Min

Determined

value

mg/100g

110.6±5.6 102.4±5.8 98.1±6.1 83.4±6.6

Corrected

value

mg/100g

114.5±5.8 106.1±6.0 101.6±6.3 86.4±6.8

The obtained results are listed in Table 2. It can be

seen that the AA concentration quite fast decreased in the surface tissue of the pepper sample prepared with old stile iron blade. Much higher AA concentration was detected in sample slice prepared with the new ceramic blade, and the oxidation appeared also slow. The stainless steel blade also released much smaller amount of catalyst than the iron.

RSD values presented in Table 2 ranges between 5-5.9 % in case of the slices prepared with ceramic blade and 6.2-7.9 % in the other ones.

The technique presented here is a cheap, powerful method for the reagentless, rapid, in situ AA determination in paprika fruit. Local concentration of the analyte is detectable without bringing the

uncertainty of the sample pretreatment and a reliable data is resuted by using the recommended viscosity correction. Local changings in AA can be determined which enables the estimation of the effect of metal ions which can get into the fruit tissue derived from practically applied knives. The diffusion layer modified electrode operated with relatively long equilibration time and very short time chronoamperometric detection program, resulted in a good operational stability –slight electrode signal diminution was observed after 30 recordings-. Furthermore the differences in tortuosity of the matrix does not influence the results and does not increase the RSD values between the measurements carried out at different places.

However the limitations of the method should be taken in consideration as well. In paprika fruit the AA concentration is slightly different in diverse parts of the fruits therefore the determined values are dependent of the locality of the working electrode. The inhomogenity of the analyte is reflected in the results which increases the RSD values. By the application of this technique, only the AA content of the fruit fluid, that is the extracellular liquid can be estimated. The molecules adsorbed or being inside of the intact cells effect the extracellular concentration through different equilibriums.

3. Conclusion

Attempt was made to work out direct electrochemical measuring technique for estimation the AA content of freshly cut yellow pepper fruits without special sample treatment or separation steps. The interfering effect of tortuosity, electrode fouling and the viscosity has been investigated. Diffusion layer modified glassy carbon electrode combined with short time chronoamperometric measuring method and viscosity change correction gave acceptable results comparing it with concentration values obtained with classical iodometric titrations. It was shown that yellow pepper slices prepared with iron blade lose their AA concentration much faster than those prepared with ceramic or stainless steel blades.

4. Acknowledgements

The authors Zs. Őri, L. Nagy, L. Kiss appreciate the support of the foundations “Synthesis of supramolecular systems, examination of their physicochemical properties and their utilization for separation and sensor chemistry” (SROP-4.2.2.A-

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11/1/KONV-2012-0065). Zs Ö. thanks for Gedeon Richter Plc. financing her stipendium by the PhD scholarship program of the Talentum Fundation.

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[9] A. M. Pisoschi, A. Pop, A. I. Serban, C. Fafaneata, Electrochim Acta. 2014, 121, 443– 460

[10] R.N. Adams, Anal Chem. 1976, 48, 1126A.

[11] A. M. Pisoschi, A. Pop, G. P. Negulescu, A. Pisoschi, Molecules. 2011, 16, 1349-1365.

[12] S. Yilmaz, M. Sadikoglu, G. Saglikoglu, S. Yagmur, G. Askin, Int J Electrochem Sc. 2008, 3, 1534.

[13] W. Okiei, M. Ogunlesi, L. Azeez, V. Obakachi, M. Osunsanmi, G. Nkenchor, Int J Electrochem Sc. 2009, 4, 276.

[14] A. Tuerke, W. J. Fischer, N. Beaumont, P. A. Kilmartin, Electrochim Acta. 2012, 60, 184-192.

[15] P. A. Kilmartin, A. Martinez, P. N. Bartlett, Curr Appl Phys. 2008, 8, 320.

[16] F. Kivlehan, F. Garay, J. D. Guo, E. Chaum, E. Lindner, Anal Chem. 2012, 84, 7670-7676.

[17] F. Rainey, K. Felynncia, C. Francine, E. Chaum, E. Lindner, Electroanal. 2014, 26, 1295-1303.

[18] L. Kiss, Zs. Ori, L. Nagy, B. Kovács, G. Nagy, J Solid State Electr. 2013, 17, 3039-3045.

[19] C. D. Kroenke, J. J. Neil, J. Neurochem Int. 2004, 45, 561–568.

[20] L. Kiss, B. Kovács, G. Nagy, J Solid State Electr. 2014 Article in press.

[21] Y. Wahyuni, A. R. Ballester, E. Sudarmonowati, R. J. Bino, A.G. Bovy, J Nat Prod. 2013, 76, 783-793.

[22] A. Morales-Soto, A. M. Gomez-Caravaca, P. Garcia-Salas, A. Seguaro-Carreto, A. Fernandez-Gutierrez, Food Res Int. 2013, 51, 977-984.

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[24] M. T. Tarrago-Trani, K. M. Phillips, M. Cotty, J Food Compos Anal. 2012, 26, 12-25.

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Direct voltammetric determination of ascorbic acid in natural paprika fruits without sample pretreatment

Zsuzsanna Őri,a,b Lívia Nagy,a László Kiss,a Barna Kovács,b Géza Nagy b* a University of Pécs, Szentágothai János Research Center, Pécs, Ifjúság útja 20, 7624, Hungary b University of Pécs, Department of General and Physical Chemistry, Ifjúság útja 6, H-7624 Pécs, Hungary * e-mail: g-nagy@gamma.ttk.pte.hu

Received: ((will be filled in by the editorial sttaff)) Accepted: ((will be filled in by the editorial sttaff))

Abstract Direct electrochemical measuring technique for estimation the ascorbic acid (AA) content of freshly cut yellow pepper fruits without special sample treatment was worked out. Dialysis membrane modified glassy carbon electrode combined with short time chronoamperometric measuring method was employed to eliminate the interfering effect of tortuosity and electrode fouling. Viscosity change inside the diffusion layer was taken in account. AA content of the fruit was found to be 114.5 mg/ 100g, and it is shown that the metal ions derived from iron blade are able to accelerate the oxidation process significantly. Classical iodometric titration was applied as a reference method.

Keywords: Ascorbic acid, Paprika, Tortuosity, Vitamins, Voltammetry

DOI: 10.1002/elan.((will be filled in by the editorial sttaff))

1. Introduction

Ascorbic acid (AA) is a small-molecular-weight, water-soluble antioxidant vitamin that participates in many biochemical reactions [1] and serves as a quality indicator of foods and drinks [2]. The quick monitoring of vitamin C levels during food production and quality control stages is an important analytical task [3]. Electrochemical techniques are often preferred to laborious instrumental methods [4, 5, 6, 7, 8] in AA assessment due to the simplicity of the procedure and of the instrumentation, to the fast response, and low cost. [9] Ascorbic acid can be easily oxidized to dehydroascorbic acid (DHA), and this redox reaction is used for its electrochemical determination [10].

Several examples of viable AA determination in various media by bare electrodes can be found in the literature, even in the presence of interferents. These methods include sample pretreatment like centrifugation [11, 12] or filtration and dilution [13] in case of fruit juices. The build-up of oxidation products can diminish the electrode response, requiring lengthy electrode cleaning procedures between each measurements [14]. PEDOT conducting polymer modified gold electrode was prepared by Türke et al.

[14], and tested by CV measurements. The peak related to the AA oxidation was obtained at 240 mV potential, was well separated from the main polyphenol oxidation peak in model wine however, repetitive analysis of AA determination in real sample with the same electrode was not carried out [14]. Thin films of polyaniline containing dopant ions polyvinylsulfonate (PVS) and polystyrenesulfonate (PSS) have been prepared on Pt disk microelectrodes and tested for AA oxidation. A potential of 0.1 V (v.s. SCE) was found to be appropriate for the amperometric determination of AA in beverages with minimal interference from other oxidisable compounds. However, exposure to wine and orange juice led to a diminution of the sensor response. The electrode fouling was due to adsorption of some beverage components [15]. A promising solution for the elimination of the electrode fouling and interference was presented for an electroactive anesthetic drug, propofol, determination in a complex medium. The method employs a PVC membrane coated GC working electrode [16, 17].

“Built-in diffusion layer” modified GC working electrode with chronoamperometric detection was developed recently for analyte determination in tortuous medium [18]. If the sample is a tortuous

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suspension containing particulate matter, live or fragmented tissue , pulp, etc., the mass transport of electroactive species in it is hindered [19]. In voltammetric analysis, for assessing the electroactive analyte concentration, the current value taken in sample solution is compared to current values recorded in calibrating standards. The voltammetric current depends on the mass transport characteristics of the analyte in the investigated sample matrix. Therefore, in order to get reliable sample concentration value, the mass transport properties must be identical in the calibrating solutions and in real samples. As long as the diffusion profile of the analyte is inside of the built-in diffusion layer, the calibrating chronoamperometric transients can be used for evaluating transients (at each identical time intervals) taken in tortuous media due to the fact that the sample tortuosity has no effect on diffusion coefficient of the analyte in the built-in diffusion layer. It is identical in both cases regardless the difference in tortuosity of the media. The applicability of the in situ analytical method was tested for determination of active chlorine concentration in different bleaching powder treated soil samples [20].

Capsicum species (pepper/paprika) are grown all over the world, primarily in tropical and subtropical countries and the fruits are an excellent sources of health-related compounds, such as ascorbic acid (vitamin C), carotenoids, tocopherols, capsaicinoids and flavonoids [21]. A comprehensive characterization of the phenolic and other polar compounds were performed by HPLC-MS method in pepper samples and besides the two identified isomers of ascorbic acid, 43 further compounds were found. Among them, AA has almost the smallest molecular weight [22].

Recently, a Screen-Printed Carbon electrode modified with Nylon-6 nanofibers membrane was successfully applied for amperometric determination of AA in fruits without sample treatment. The membrane significantly improved the selectivity towards AA, and entailed a widening of the linearity range [23]. In our study, a dialysis membrane modified GC electrode with short time chronoamperometric detection is applied for the reagentless, in situ assessment of the AA concentration in paprika fruit without sample pretreatment. A resting period with opened circuit prior to the measurement enables the equilibration of the membrane layer by the analyte, and the depletion of the remaining oxidation products. Due to the appropriately short time detection the tortuosity does not affect the result–as the diffusion profile is still inside the membrane layer during the

measurement-, and there is a minimal possibility for the electrode fouling. Therefore the presented method is presumed to possess further advantages in comparison to the methods employing conventional amperometric detection technique.

Traces of iron can get into the fruit tissue during food processing. Using our method the catalytic effect of this can be detected. The extent of the autooxidation depends on the nature of the cutting blade used for slicing the fruit tissue.

2. Experimental

Chemicals and materials

Ascorbic acid (AA), sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, glycerol, potassium iodide, iodine, soluble starch used were of analytical reagents grade and were purchased from VWR BDH Prolabo; while iodine and potassium chloride were obtained from Reanal (Hungary).

Phosphate buffer made with double deionized water and containing 0.5M KCl served as solvent for AA solutions and was used as background electrolyte for measurements in aqueous solutions. The pH of the buffer was adjusted to 5.9 in order to match the natural pH of the paprika juice. It worth mentioning that at low pH AA is fully protonated and is relatively stable. Its maximal stability was found in the pH range of 4–6 [24]. AA concentration of different solution is expressed throughout the text in mg/100 g unit in accordance with the generally applied practice in food sciences. AA solutions were prepared each time freshly before use. In all cases -except for the direct measurements in the paprika tissue- dissolved oxygen was purged out from the cell by bubbling high purity nitrogen for 10 min before the experiment.

Glassy carbon (GC) rod for electrode preparation was purchased from VWR International Ltd. Buehler (USA). MicroPolish alumina powder of different particles size and Buehler MicroCloth (No. 40–7218) were used in wet electrode polishing. The dialysis membrane (Technikon TM Pre-mount type “C”) used as size-exclusion diffusion layer was taken from AutoAnalyzer accessories (Technicon Instruments Corporation) with a thickness of 127 µm; pore diameters ranged between 4 and 6 nm.

Yellow sweet pepper (Capsicum annuum Linn.) was purchased from the local supermarket, and it was washed under tap water. For juice preparation the peppers were masticated with a plastic rasp and the

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obtained liquid was separated filtering it through a plastic kitchen strainer. In case of making pepper slice samples ceramic-, stainless steel- or iron blade was used to break up cells slightly on the surface of the paprika tissue. The blades were in contact with the paprika tissue for about 2-3 minutes.

For viscosity measurements paprika juice was centrifuged with 10.000 rpm for 5 min then the transparent aliquot was filtered with a 0.2 micron Injection Filter (VWR BDH Prolabo). AA solutions of different viscosity were made mixing buffer solution and glycerol in different ratio as follows: 150mg AA dissolved in 10 ml buffer; 150 mg AA in 9.75ml buffer + 0.25ml glycerol; 150 mg AA in 9.5ml buffer + 0.5ml glycerol and the same amount of solute in 8.75ml buffer + 1.25ml glycerol.

The iodine reagent used in titrations was made dissolving 2 g potassium iodide and 1.3 g iodine in 1dm3 water.

Electrodes

Homemade GC disc electrode (3 mm in diameter) was used as working electrode sealed in cylindrical Teflon body, a Pt plate as counter electrode (∼4 cm2) and Saturated calomel electrode (SCE) as reference one were employed. The surface of the GC electrode was wet polished with 1, 0.3, and finally, 0.05-µm particle size alumina polishing powder, washed and sonicated for 1 min between and after each polishing steps. Diffusion layer modified GC electrode was made by coating the measuring surface of a freshly polished wet GC electrode with a dialysis membrane cup. It was fixed using a thread and great care was taken to avoid air bubbles under the membrane.

Instruments and electrochemical measurements

In the cyclic voltammetric and chronoamperometric studies, conventional beaker-type measuring cell was applied, except when the chronoamperometric measurements carried out directly on paprika slices. In all measurements CHI 760C electrochemical workstation (CH Instruments, Inc. USA) was employed.

After each chronoamperogram recording the electric circuit was opened to allow time to reach concentration equilibrium between the sample and the diffusion layer. The required time for this equilibration process was determined experimentally. Sampling interval was set to 0.01 s.

Appropriate electrode potential was selected by recording linear sweep voltammograms previously. All measurements were carried out at room temperature. All electrode potentials were measured vs. saturated calomel reference electrode (SCE). The chronoamperometric measurements were performed at 0.6 V constant potential vs. the reference.

Viscosity measurements were carried out at controlled temperature (22 oC) with the application of a Capillary Viscosimeter (Cannon-Fenske Routine, Fungilab ASTM D-445) and the liquid density was determined with a Pycnometer (4AJ-9277400).

Direct electrochemical measurement in paprika

In these experiments the yellow paprika fruits were cut to slices, the surface of the internal side of the slices were slightly ruptured by blades, in 1-2 millimeters depth. The three electrodes were held by one electrode positioner, keeping constantly 2-3 mm distances between the electrode bodies. The working electrode was placed between the counter and the reference one. Before the measurements the electrodes were slowly brought in touch with the wet surface making sure that the membrane of the working electrode is not compressed by the weight of the electrode so the porosity of it is not changed. The counter electrode was gently pricked into the tissue. The scheme of the direct measurement in the fruit is shown in Fig. 1.

Fig. 1. Scheme of the direct measurement carried out in paprika slice. WE: dialysis membrane coated GC electrode, CE: Platinum sheet counter electrode, RE: Saturated calomel electrode.

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2. Results and discussion

As it is well known AA is electroactive. In buffered solution it gives well formed voltammograms using different electrodes and measuring techniques. Assessment of AA concentration in aqueous solution can be done quite easily employing voltammetric methods. The juice of fruits or vegetables can be good background electrolyte for voltammetric measurements owing to their relatively high electrolyte concentration and more or less buffered pH. Therefore it was expected that AA content of vegetables or fruits could be estimated recording voltammograms directly with a special working electrode poked inside their tissue or immersed into the juice prepared by simply crushing their tissue.

Trying to work out voltammetric method for measuring AA in yellow pepper directly, without sample pretreatment or separation step we prepared juice from paprika samples and recorded cyclic voltammograms one after the other in quiescent solution with 0.1 V/s scanning rate using GC working electrode. Between the voltammograms 60 s was the waiting period. Well-formed peak shaped voltammograms were obtained indicating that the juice contained AA. Similar measurements were made in buffered AA solution containing AA in concentration of 100mg/100g. The peak heights (ip) of the voltammograms were measured. The first one was disregarded but the others were normalized to the ip of the second voltammogram (ip2). The obtained ip/ip2. ratios were plotted against the serial number of the sequential voltammograms. The same experiments were made with dialysis membrane coated electrode. The obtained dependences are shown in Fig. 2.

Fig. 2. Normalized peak currents of sequentially recorded cyclic voltammograms recorded with (■) bare GC and with (●) membrane coated GC electrode in buffered AA solution (100mg/ 100g) Normalized peak currents of sequentially recorded cyclic voltammograms recorded with (▲) bare GC and with (▼) membrane coated GC electrode in paprika juice. Potential window: 0-0.9V vs. SCE, Scanning rate: 0.1V/s. Quiet time before and between the CV-s was 60 seconds.

It can be seen that that the peak heights of the

voltammograms taken in buffered AA solution are almost the same. In the tested juice, however the height of the voltammetric peaks recorded with the bare electrode steadily decreases. This indicates the fouling of the electrode. Some ingredient of the juice upon taking part in electrode reaction decreases steadily the activity of the glassy carbon surface. The tenth ip obtained in paprika juice medium is smaller than 75% of the second one. This sensitivity loss caused by electrode fouling seriously obstructs the application of direct voltammetric measurement for quantification of AA content of the juice. Much less electrode fouling could be observed when the voltammograms were made with the membrane coated electrode. Most likely the dialysis membrane acts like a size exclusion layer. It decreases the transport of the harmful species to the electrode surface.

In case of cyclic voltammetric measurements the oxidizing electrode potential (range) is applied for relatively long time periods. Long-time oxidizing potential exposure results in extensive fouling. Applying short time chronoamperometric measurements at the appropriate electrode potential the measuring time can be shortened drastically. We can expect that using dialysis membrane coated electrode and short time measurement with chronoamperometric technique the interference with direct AA assessments in pepper juice caused by electrode fouling can be dramatically decreased.

There is another difficulty if working electrode directly poked into the pepper tissue is tried to be used for measurement of AA content of the natural tissue. It is the tortuosity. The voltammetric measurements generally rely on diffusion of the electroactive species. In tortuous tissue the diffusion of the AA is slower than it is in aqueous solution. Therefore the evaluation of the voltammetric data taken in the tissue cannot be done simply using calibration curves obtained in aqueous solution. In case of direct measurement in tissue, the standard addition technique also cannot be employed. In our earlier experiments carried out with sediments [18] it was shown however, that using

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working electrode modified with built in diffusion layer and short time chronoamperometric measurements in tortuous media, reliable concentration values could be obtained for concentration of electroactive species. As long as the diffusion profile created by the electrode reaction stays inside the built in diffusion layer, the calibration data obtained in aqueous solution can be used for evaluating the data taken in the tortuous media.

During chronoamperometric measurements the concentration of the detected species decreases at the electrode surface. If the electrode holds a built in diffusion layer then certain time is needed between measurements for equilibration of the concentrations between the sample and the modifying layer. In order to determine the necessary equilibrating time sequential chronoamps with different equilibrating (resting) times between measurements were recorded in quiescent AA solution with the membrane covered GC electrode. The equilibration period was set to 15, 30, 45 and 90 s long and applied before and between the repetitive measurements. The normalized current values of the short time chronoamps taken at 0.3 seconds after the electrode potential jump referring to the different equilibration time periods are shown in Fig. 3.

Fig. 3. Normalized current values taken at 0.3 s of the sequentially repeated chronamps using different equilibration time periods between measurements. The current values referring to the repetitions are normalized to the current values of the second chronoamps (i / i2). Dialysis membrane coated GC electrode was used and the AA concentration of the buffered solution (pH 5.9) was 100 mg/ 100g. The equilibration period was set to be 15, 30, 45 and 90 s long with opened circuit and applied before and between the repetitive recordings. Symbols indicate the serial number of the sequentially taken i300 ms values.

As it can be seen in Fig. 3, in case of 15 s equilibration time the current value of the tenth recording is only 86% of the second one. It is clearly visible that longer the resting time the points are getting closer to each other. At the equilibration time of 90 seconds the value of the last point is approx. 100% of the second one so the points are very close to each other. In the further experiments 90 seconds resting time was applied for the equilibration in quiescent medium.

From the experimental findings shown before we could see that, if a working electrode modified with a built in diffusion layer and short time chronoamperometric measurements with long enough equilibrating time is employed, then the AA concentration of yellow pepper tissue could be assessed directly without sample pretreatment and separation steps. The interfering effects of sample tortuosity and matrix caused electrode fouling can be reduced to the acceptable level. However there is one more property of the sample that can bring in uncertainty. It is the viscosity.

In chronoamperometry the current depends on the diffusion coefficient of the electroactive species under diffusion mass transport controlled conditions. Diffusion coefficient depends on the dynamic viscosity. Matrix components can penetrate into the diffusion layer and can change the viscosity there. This effect can question the reliability of the calibration data taken in aqueous solutions.

In order to study the potential effect of viscosity change on the short time chronoamperometric current transients model solutions of different viscosity containing 150mg/ 100 ml AA were made. The solvent of these solutions were prepared by mixing PBS buffer and glycerol in the following V/V % ratios: 87.5/12.5; 95/5; 97.5/2.5; 100/0.

Chronoamperometric measurements of AA oxidation recorded in differently viscous solvent mixtures are shown in Fig. 4.

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Fig. 4. Chronoamperometric transients of AA oxidation recorded in solvent mixtures of different viscosity. Model solutions were composed of 150mg / 100 ml AA and solvent of aq. PBS buffer and of glycerol in varied ratio: d- 87.5/12.5; c- 95/5; b- 97.5/2.5; a- 100/0. (V/V % aq. buffer/V/V% glycerol) Measuring potential: 0.6V vs. SCE, Working electrode: bare GC.

As it can be seen in Fig. 4, chronoamps run at gradually lower and lower current values in accordance with the increasing ratio of glycerol in the solvent mixtures.

If the rate determining step of electrode process is the linear diffusion then after an appropriate potential step the chronoamperometric curves are described by the Cottrell equation,

� ������

��

�����

(1)

Where: i is the current; n: number of electrons; F: Faraday constant; A: surface area of the electrode; cj0: initial concentration of the analyte; D: diffusion constant; t: time. And since, the diffusion coefficient should vary with the viscosity, according to the Stokes-Einstein equation,

� ���

� �� (2)

-where: k: Boltzmann constant; T: absolute temperature; a: effective radius of the molecule-, η is the viscosity then linear relation between i and η-1/2

should hold if the effective radius is constant in the studied range of the examined solvent composition. A plot of inorm versus η-1/2 shows the experimental data in

Fig. 5. Inorm values refer to the chronoamps presented in Fig. 5. and are normalized to that of the current values taken in 100% aq. buffered AA solution. The hydrodynamic viscosity values of the model solutions were determined by using a capillary viscometer and a picnometer.

Since the aim of this experiment is to estimate the deviation in the current due to viscosity discrepancy between the aq. AA solution and the penetrating paprika juice fluid, dynamic viscosity of the centrifuged, filtered paprika juice was measured at the same way like in case of the glycerol-buffer model solutions. The obtained value is indicated in Fig. 5. Calculated inorm value for the centrifuged, filtered Paprika juice is concluded from the experimentally obtained η value for this sample.

Fig. 5. Experimentally obtained inorm current values versus the reciprocal square root of the measured η. Model solutions were composed of 150mg/ 100 ml AA and solvent of aq. PBS buffer and of glycerol in varied ratio: d- 87.5/12.5; c- 95/5; b- 97.5/2.5; a- 100/0. (V/V % aq. buffer/V/V% glycerol). Inorm values are the normalized currents to that of the 100% aq. buffered solution. (inorm =(i/ia)x100) Calculated inorm value for the centrifuged, filtered paprika juice is concluded from the experimentally obtained η value for this sample.

As it can be seen in Fig. 5, the points of the model solutions lay on a straight line with an adjusted R2= 0.996 value so the assumption on the basis of the two equations is justified under these experimental conditions.

Regarding the experimentally obtained η= 1.111 mPa*s dynamic viscosity value for the centrifuged, filtered paprika juice compared to that of the of the 100% buffered AA solution (η=1.011mPa*s), on the basis of the justified correlation, the calculated current value for the paprika fluid is estimated to be 96.36% of the 100% aq. buffered AA solution, that means if the effective radius of the analyte molecules is considered

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to be the same in these systems as well, approx. 3.6% current decreasing is expected due to the viscosity differences.

In order to test the analytical performance and applicability of the diffusion layer coated electrode in the paprika juice - keeping the identified parameters: 90 s equilibration time, 0.3 s-long chronoamp measuring period- standard addition experiments were carried out in the unfiltered paprika juice and in 100 mg/ 100 g AA solution in a parallel way.

During the chronoamperometrically followed standard addition experiments, 20-20-µl of 1000 mg AA/ 5 ml PBS buffer stock solution was spiked repeatedly into 10 ml of the sample solution by a Hamilton-syringe. The sample solution was purged for ten minutes by water saturated nitrogen flow prior and between the measurements. Each addition steps resulted in concentration increase of 40 mg/ 100 g (neglecting the dilution by the solvent dose). After a chronoamperometric measurement, the electrode was removed from the solution, the sample was spiked by a dose, then 30 sec stirring was applied and finally the electrode was immersed into the bulk again.

Fig. 6b shows the results for the described series of measurements taken by the membrane modified electrode while Fig. 6a is a plot for presenting the same experiments under the same conditions carried out with an uncoated GC electrode.

Fig. 6. Standard addition experiments carried out parallel in 100mg AA/ 100g PBS solution and in unfiltered Paprika juice. a) chronoamperometric measurements taken by uncoated bare GC electrode b) chronoamperometric measurements taken by the dialysis membrane coated GC electrode.

As a reference method, redox titration using iodine solution was used to compare the results obtained with the new electrochemical method. In this case 10 ml of the sample solution (AA solution or Paprika juice appropriately spiked) was titrated with iodine solution in a conical flask containing about 75 ml of distilled water and 1 ml of starch indicator solution. Comparison of the results of the standard addition experiments are summarized in Table 1.

Table 1. Summary of the results of the standard addition experiments carried out in unfiltered Paprika juice by three different methods: chronoamperometry with bare GC electrode, chronoamperometry with dialysis membrane layer coated electrode and redox titration).

Bare GC,

ampero.

Modified GC,

ampero.

Redox

titration

Linearity Tendentious

deflection Adj.R

2=0.999 Adj.R

2=0.996

Recovery

% ---

89.5

93.1 # 93.9

Original

AA conc.

mg/100g

--- 115.9 115.5

[#] with viscosity correction

The suggested chronoamperometric method comparing to the titration, resulted in quite accurately determined AA value and outstanding linearity. The recovery value of the chronoamperometric method applying the membrane coated electrode is 4.3% lower

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than that of the titration method. This difference exists mainly due to the investigated viscosity difference between the aquaeous solution and the paprika fluid that was calculated to be approx. 3.6%. So in the future experiments a correction factor is recommended in the evaluation of the chronoamperometric measurements with the membrane coated electrode carried out in the same matrix. The remaining 6-7% recovery loss probably reflects the intrinsic matrix effect of the unfiltered paprika juice.

The method worked out was used for determination of AA in yellow pepper slices prepared with different knifes. In these studies the intact yellow pepper fruits were cut to slices by three different knifes, namely with ceramic, stainless steel and old style iron blade. The surface of internal side of the sample slices were slightly ruptured by the blades. They were kept in humid atmosphere in a beaker closed with watch glass. After a while the electrodes were brought in touch with the wet surface of the slice as it is shown in Fig 1, and upon proper equilibrium time (90s) chronoamps were recorded and the AA concentration was estimated using calibration data taken in aqueous solutions.

Table 2. The analytical results indicating the rate of oxidation of AA content of differently prepared yellow pepper slices. ∆t value indicates the time periods between the application of the blades and the measurements.

Ceramic

blade

∆t=0-10

min

Ceramic

blade

∆t=60-80

min

S. steel

blade

∆t=60-80

Min

Iron

blade

∆t=60-80

Min

Determined

value

mg/100g

110.6±5.6 102.4±5.8 98.1±6.1 83.4±6.6

Corrected

value

mg/100g

114.5±5.8 106.1±6.0 101.6±6.3 86.4±6.8

The obtained results are listed in Table 2. It can be

seen that the AA concentration quite fast decreased in the surface tissue of the pepper sample prepared with old stile iron blade. Much higher AA concentration was detected in sample slice prepared with the new ceramic blade, and the oxidation appeared also slow. The stainless steel blade also released much smaller amount of catalyst than the iron.

RSD values presented in Table 2 ranges between 5-5.9 % in case of the slices prepared with ceramic blade and 6.2-7.9 % in the other ones.

The technique presented here is a cheap, powerful method for the reagentless, rapid, in situ AA determination in paprika fruit. Local concentration of the analyte is detectable without bringing the

uncertainty of the sample pretreatment and a reliable data is resuted by using the recommended viscosity correction. Local changings in AA can be determined which enables the estimation of the effect of metal ions which can get into the fruit tissue derived from practically applied knives. The diffusion layer modified electrode operated with relatively long equilibration time and very short time chronoamperometric detection program, resulted in a good operational stability –slight electrode signal diminution was observed after 30 recordings-. Furthermore the differences in tortuosity of the matrix does not influence the results and does not increase the RSD values between the measurements carried out at different places.

However the limitations of the method should be taken in consideration as well. In paprika fruit the AA concentration is slightly different in diverse parts of the fruits therefore the determined values are dependent of the locality of the working electrode. The inhomogenity of the analyte is reflected in the results which increases the RSD values. By the application of this technique, only the AA content of the fruit fluid, that is the extracellular liquid can be estimated. The molecules adsorbed or being inside of the intact cells effect the extracellular concentration through different equilibriums.

3. Conclusion

Attempt was made to work out direct electrochemical measuring technique for estimation the AA content of freshly cut yellow pepper fruits without special sample treatment or separation steps. The interfering effect of tortuosity, electrode fouling and the viscosity has been investigated. Diffusion layer modified glassy carbon electrode combined with short time chronoamperometric measuring method and viscosity change correction gave acceptable results comparing it with concentration values obtained with classical iodometric titrations. It was shown that yellow pepper slices prepared with iron blade lose their AA concentration much faster than those prepared with ceramic or stainless steel blades.

4. Acknowledgements

The authors Zs. Őri, L. Nagy, L. Kiss appreciate the support of the foundations “Synthesis of supramolecular systems, examination of their physicochemical properties and their utilization for separation and sensor chemistry” (SROP-4.2.2.A-

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11/1/KONV-2012-0065). Zs Ö. thanks for Gedeon Richter Plc. financing her stipendium by the PhD scholarship program of the Talentum Fundation.

4. References

[1] B. Y. Beker, I. Sönmezoglu, F. Imer, R. Apak, Int J Food Sci Nutr. 2011, 62(5), 504-512.

[2] C. V. Popa, A. F. Danet, S. Jipa, T. Zaharescu, Rev Chim-Bucharest. 2012, 63, 978.

[3] P. F. Hsu, W. L. Ciou, P. Y. Chen, J Appl Electrochem. 2008, 38, 1285.

[4] D. A. Skoog, D. M. West, F. J. Holler. Fundamentals of Analytical Chemistry, 7th ed., Saunder College Publishing, Philadelphia, 1998, p. 845.

[5] J. Borowski, A. Szajdek, E. J. Borowska, E. Ciska, H. Zielinski, Eur Food Res Technol. 2008, 226, 459.

[6] M. Rodriguez-Comesa˜na, M. S. Garcia-Falcon, J. Simal-Gandara, Food Chem. 2002, 79, 141.

[7] S. Vermeir, M. L. Hertog, A. Schenk, K. Beullens, B.M. Nicolai, J. Lammertyn, Anal Chim Acta. 2008, 618, 94.

[8] K. Guclu, K. Sozgen, E. Tutem, M. Ozyurek, R. Apak, Talanta. 2005, 66, 1226.

[9] A. M. Pisoschi, A. Pop, A. I. Serban, C. Fafaneata, Electrochim Acta. 2014, 121, 443– 460

[10] R.N. Adams, Anal Chem. 1976, 48, 1126A.

[11] A. M. Pisoschi, A. Pop, G. P. Negulescu, A. Pisoschi, Molecules. 2011, 16, 1349-1365.

[12] S. Yilmaz, M. Sadikoglu, G. Saglikoglu, S. Yagmur, G. Askin, Int J Electrochem Sc. 2008, 3, 1534.

[13] W. Okiei, M. Ogunlesi, L. Azeez, V. Obakachi, M. Osunsanmi, G. Nkenchor, Int J Electrochem Sc. 2009, 4, 276.

[14] A. Tuerke, W. J. Fischer, N. Beaumont, P. A. Kilmartin, Electrochim Acta. 2012, 60, 184-192.

[15] P. A. Kilmartin, A. Martinez, P. N. Bartlett, Curr Appl Phys. 2008, 8, 320.

[16] F. Kivlehan, F. Garay, J. D. Guo, E. Chaum, E. Lindner, Anal Chem. 2012, 84, 7670-7676.

[17] F. Rainey, K. Felynncia, C. Francine, E. Chaum, E. Lindner, Electroanal. 2014, 26, 1295-1303.

[18] L. Kiss, Zs. Ori, L. Nagy, B. Kovács, G. Nagy, J Solid State Electr. 2013, 17, 3039-3045.

[19] C. D. Kroenke, J. J. Neil, J. Neurochem Int. 2004, 45, 561–568.

[20] L. Kiss, B. Kovács, G. Nagy, J Solid State Electr. 2014 Article in press.

[21] Y. Wahyuni, A. R. Ballester, E. Sudarmonowati, R. J. Bino, A.G. Bovy, J Nat Prod. 2013, 76, 783-793.

[22] A. Morales-Soto, A. M. Gomez-Caravaca, P. Garcia-Salas, A. Seguaro-Carreto, A. Fernandez-Gutierrez, Food Res Int. 2013, 51, 977-984.

[23] C. A. Fuenmayor, S. Benedetti, A. Pellicanó, M. S. Cosio, S. Mannino, Electroanal. 2014, 26, 704-710.

[24] M. T. Tarrago-Trani, K. M. Phillips, M. Cotty, J Food Compos Anal. 2012, 26, 12-25.

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Response to decision letter:

Dear Professor Wang, Dear Reviewers,

Thank you for your further consideration of the manuscript. Obtaining the referees opinion about the

paper we corrected the manuscript closely following the helpful suggestions and a few minor changes

have also been introduced. The authors hope that all the queries of the Reviewers have been addressed.

Hereby we submit our paper hoping that in this revised form the manuscript meets the standards of

Electroanalysis.

Response to comments of Reviewer 1:

“The authors report a method for the detrmination of ascobic acid in paprica fruit without taking in

account the most recent literature ( see Fuenmayor et al . 2014 Electroanalysis 26(4), 704-710). So

nothing new from the analyical point of view and electrode preparation.”

We are sorry that we missed the mentioned paper. It appeared recently in 2014. We have been working on

application of electrodes with built in diffusion layer for analysis in tortuous media. This task goes back to

the time of in vivo measurements of neurotransmitter molecules. Then working in the laboratory of Ralf

Adam (G. Nagy) it was questioned how fare the calibration data recorded in aqueous solutions could be

used for estimation of the local concentration in the brain of anesthetized experimental animals.

The cited publication is added to the introduction and references and evaluated as it follows:“Recently, a

Screen-Printed Carbon electrode modified with Nylon-6 nanofibers membrane was successfully applied

for amperometric determination of AA in fruits without sample treatment. The membrane significantly

improved the selectivity towards AA, and entailed a widening of the linearity range.”

There is no question that similarities between the published work of Fuenmayor et al. and the presented

work exist, like the application of a membrane layer for improvement of the selectivity and electrode

damage prevention. However, there is an additional function of the membrane layer in our case –it serves

as a built-in diffusion layer as well- and the detecting technique is different. A resting period with opened

circuit prior to the measurement enables the equilibration of the membrane layer by the analyte, and the

depletion of the remaining oxidation products. Due to the appropriately short time detection the tortuosity

does not affect the result–as the diffusion profile is still inside the membrane layer during the

measurement-, and there is a short oxidation time reducing or even avoiding the electrode fouling.

Therefore the presented method is presumed to possess further advantages in comparison to the methods

employing conventional amperometric detection technique.

A newly studied aspect of our work relates to the viscosity of the fruit fluid that can be slightly different

from that of the calibrating aqueous solutions. The effect of it was taken in account since the currents are

dependent of the dynamic viscosity as it is given by the Cottrell and Stokes-Einstein equations.

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As far as the authors know the local decrease in ascorbic acid concentration catalyzed by metal ions

derived from different kind of blades has not been investigated however it has a significant practical

importance.

“Furtemore, by using an old reference method based on iodine titration, well know to be not precise or

accurate, make the result aimless. What's new? what is the difference? CLARIFY!”

Redox titration can be applied to determine the ascorbic acid concentration directly in the paprika juice

containing pulp in order to compare the data obtained using the method recommended by us.

The results of the methods can be compared without bringing the uncertainity of the sample handling or

extraction. Of course there are modern methods which probably give more accurate results than the

proposed one, in case of the correct sample handling and pretreatment. However our goal is to develop a

rapid method useful in direct measurements.

We hope that we could clarify that the short term detection technique and the application of working

electrode with built in diffusion layer has some advantage as well as novelty that could be applied in

different samples with tortuous matrix that the standard addition technique cannot be applied.

Response to comments of Reviewer 2

“The Determination of ascorbic acid is of great importance in Food science. This manuscript adresses this

Problem by describing an EC method based on a GC electrode coated with a Membrane to quantify

ascorbic acid in Paprika Juice. Also, the application of the sensor directly in the Paprika tissue is

presented.

One of the merit of this work is that it takes into account several factors which normally hinder the

electrochemical detection of ascorbic acid in real matrices. Resting time, electrode fouling and viscosity

are well discussed. In addition, the final application on real Paprika sample is also original.

Through the reading of the text, I noticed only minor flaws which require some more attentions. The main

concern is on the problem of oxygen dissolved in the experimental solutions. At PAge 2 Line 18, Column

right it is reported that: "In some experiments the dissolved oxygen was purged out ..." This is not clear.

The Authors should always report when the bubling was used. The reason is that AA reacts very fast with

Oxygen dissolved in the solution and in the air above the solution. This can prejudice the Quality of the

data reported. Please, explain how did you control the presene of Oxygen during the analysis.”

We were glad to learn that our referee finds the work valuable and suggests only helpful changes. As fare

as we could we made the recommended changes and additions.

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At the indicated part the sentence was corrected: “In all cases -except for the direct measurements in the

paprika tissue- dissolved oxygen was purged out from the cell by bubbling high purity nitrogen for 10 min

before the experiment.” In case of the direct measurements carried out on paprika slices the exclusion of

the atmospheric oxygen was not performed, since the purpose of the investigation is to detect the local

ascorbic acid concentration in fruit slices as it is changed during conventional food processing.

“The same description should be given for the description of the standard addition method (Page 6 Line

53, column left). These experiment should be performed under Nitrogen flow. Otherwise, AA will react

with the Oxygen dissolved in the solution and with the Oxygen in the head space.”

At the indicated part the following sentence was inserted: “The sample solution was purged for ten

minutes by water saturated nitrogen flow prior and between the measurements.”

“At Page 7 Line 50 column left, it is important to explain better the method used and discuss its

limitations. How did you positioned the three electrodes on the surface of the fruit ? Also, how did you

control the distance between the three electrodes? The Experiment should be better explained. Consider to

add a dedicated section in the Experimental.”

In the Experimental section a dedicated section is inserted for the better presentation of the electrode

arrangement and positioning as it follows:“The three electrodes were held by one electrode positioner,

keeping constantly 2-3 mm distances between the electrode bodies. The working electrode was placed

between the counter and the reference one. Before the measurements the electrodes were slowly brought

in touch with the wet surface making sure that the membrane of the working electrode is not compressed

by the weight of the electrode so the porosity of it is not changed. The counter electrode was gently

pricked into the tissue.”

“Also, it should be made clear that AA in the fruit is not homogeneously distributed. Thus, the place

where the measurement was taken affects the final result. Places in the fruit close to the peel have different

AA concentration than those close to the Pulp. The measurements of AA directly in the fruit may reflect

differences which are dependent on the place where the sensors were positioned.”

In the results and experiments part the reasonably questioned part is inserted as it follows:“In paprika

fruit the AA concentration is slightly different in diverse parts of the fruits therefore the determined values

are dependent of the locality and as the electrodes are replaced, the inhomogenity of the analyte is

reflected in the results which increases the RSD values.”

“A better discussion of the pro and cons of the technique is recommened.”

At the end of the results and discussion, a section dedicated to concluding the advantages and

disadvantages is introduced.

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